Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2007

- 1 -

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006

Supporting Information

for

A New Protein Engineering Approach Combining Chemistry and Biology,

Part II: Site-Specific Functionalization of Proteins by Organopalladium

Reactions

Koichiro Kodama, Seketsu Fukuzawa,* Hiroshi Nakayama, Kensaku Sakamoto,

Takanori Kigawa, Takashi Yabuki, Natsuko Matsuda, Mikako Shirouzu, Koji Takio,

Shigeyuki Yokoyama,* and Kazuo Tachibana*

Figure S1. Detection of the biotinylated Ras protein using A) Streptavidin-HRP and B) an anti-Ras antibody. The iF32- and iF174-Ras-His proteins were subjected to the Mizoroki-Heck reaction, purified using monomeric avidin beads, and analyzed by Western blot. The Y32-Ras-His protein was used as a control. An asterisk indicates a cross reaction between avidin and Streptavidin-HRP (or an anti-Ras antibody).

- 2 -

Figure S2. Electrostatic surface potentials (Left) and ribbon models (Right) of A) the GDP-bound form of the wild-type Ras protein (PDB: 4Q21)[1] and B) the GTP-bound form of the wild-type Ras protein (PDB ID: 5P21).[2] An arrow indicates tyrosine residues at position 32 and 40 of the wild-type Ras protein.

- 3 -

Figure S3. A) SDS-PAGE analysis of the wild-type Ras protein before (lane 1) and after (lane 2) being incubated under Sonogashira conditions. B) The X-Ray fluorescence (XRF) spectrum of the wild-type Ras protein (840 nM) after being incubated under Sonogashira conditions. The Pd-Lα peak (2.84 keV) was overlapped with the Ar-Kα peak (2.96 keV) derived from argon in air.

- 4 -

Scheme S1. Conjugation of the iF-peptide (3) with vinylated biotin (1) by A)Mizoroki-Heck reaction or propargylated biotin (2) by B)Sonogashira reaction.

- 5 -

Table S1. The average mass calculated from the amino acid sequence of the

Sonogashira reaction product (bF32-Ras-His).

[a] The mass value that agreed with the experimental mass value in Figure 5.

product ion (M+H)+ (M+2H)2+ product ion (M+H)+ (M+2H)2+

[m/z] [m/z] [m/z] [m/z]

b1 88.09 44.55 y25 3273.69 1637.35

b2 159.17 80.09 y24 3202.62 1601.81

b3 272.32 136.67 y23 3089.46 1545.23

b4 373.43 187.22 y22 2988.35 1494.68

b5 486.59 243.80 y21 2875.19 1438.10

b6 614.72 307.86 y20 2747.06 1374.03

b7 727.88 364.44 y19 2633.90 1317.45

b8 841.04 421.02 y18 2520.74 1260.87

b9 969.17 485.09 y17 2392.61 1196.81

b10 1083.27 542.14 y16 2278.51 1139.76

b11 1220.41 610.71 y15 2141.37 1071.19

b12 1367.59[a] 684.30 y14 1994.19[a] 997.60

b13 1466.72[a] 733.87 y13 1895.06[a] 948.03

b14 1581.81[a] 791.41 y12 1779.97[a] 890.50

b15 1710.93[a] 855.97 y11 1650.85[a] 825.93

b16 2137.47 1069.24 y10 1224.31[a] 612.66[a]

b17 2252.56 1126.78 y9 1109.22[a] 555.12

b18 2349.67[a] 1175.34 y8 1012.11 506.56

b19 2450.78 1225.89 y7 911.00 456.01

b20 2563.94[a] 1282.47 y6 797.84 399.43

b21 2693.05 1347.03 y5 668.73[a] 334.87

b22 2808.14 1404.57 y4 553.64[a] 277.32

b23 2895.22[a] 1448.11 y3 466.56 233.78 b24 3058.40 1529.70 y2 303.39 152.20

b25 3214.58 1607.80 y1 147.20 74.10

- 6 -

Experimental Section

Materials: 9-Fluorenylmethoxycarbonyl (Fmoc) and side-chain protected amino acids,

and Fmoc-CLEAR-Amide Resin were purchased from Peptide Institute (Minoo, Ja-

pan). Pd(OAc)2 (99.9999% purity) was purchased from Sojitz Chemical (Tokyo, Ja-

pan). Copper(I) trifluoromethanesulfonate (CuOTf) benzene complex, sodium ascor-

bate, and decyl-β-D-glucopyranoside (DG) were purchased from Sigma-Aldrich (St.

Louis, MO). N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) was

obtained from Dojindo (Kumamoto, Japan). Bovine serum albumin (Ultrapure BSA)

was purchased from Ambion (Austin, TX, USA). The expression plasmids pK7-Ras-

His and pK7-Ras-His-32am were gifts from Dr. Takashi Ohtsuki, Okayama University.

Ni-NTA agarose (Ni nitriloacetic acid-agarose beads) were purchased from QIAGEN

GmbH (Hilden, Germany). 12% NuPAGE® Bis-Tris gel was purchased from Invitrogen

(Carlsbad, CA, USA). The Achromobacter Protease I (Lys-C) was a gift from Prof.

Takeharu Masaki, Ibaraki University. The anti-Ras antibody (H-Ras F235) was pur-

chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The streptavidin-

horseradish peroxidase conjugate, anti-Mouse Ig (NA931V), ECL Plus Western blot

detection reagents (RPN2132), Superdex 75 (HiLoad 16/60 pg), and prepacked

disposable PD-10® Column, were purchased from GE Healthcare Bio-Sciences

(Piscataway, NJ).

Methods: An automated peptide synthesizer (PSSM-8) was a product of Shimadzu

(Kyoto, Japan). The oxygen monitor (JKO-O2LD II) was a product of JIKCO (Tokyo,

Japan). The oxygen absorber (A-500HS) was purchased from ISO (Yokohama,

Japan). The preparative HPLC system consisting of a PU980 gradient pump and a

UV980 UV-Vis detector was purchased from JASCO (Tokyo, Japan), and the Waters

600E multisolvent delivery system was purchased from Waters (Milford, MA, USA).

Low- and high-resolution mass spectra were recorded on a JEOL JMS-MS-700P

(Akishima, Japan) mass spectrometer under fast atom bombardment (FAB) condi-

tions using glycerol (positive) or triethanolamine (negative) as a matrix. The LC-MS

system was a combination of a model 1100 liquid chromatograph with a diode-array

detector manufactured by Agilent technologies (Waldbronn, Germany), and a Ther-

moelectron LCQ ion-trap mass spectrometer (San José, CA, USA). Mightysil® C18, a

reversed phase chromatographic medium, was purchased from Kanto Kagaku (Tokyo,

Japan). The Ras or biotinylated Ras band was detected with a fluorescent imaging

- 7 -

analyzer FLA2000® or LAS-3000® (Fuji Photo Film, Tokyo, Japan). The structure of

the Ras protein was displayed using the CueMol program (R. Ishitani, CueMol:

Molecular Visualization Framework; cuemol.sourceforge.jp). The X-Ray fluorescence

(XRF) analysis was performed using NANOHUNTER®, a tabletop TXRP Spectrometer

manufactured by Rigaku (Tokyo, Japan).

Preparation of iF-peptide: The tripeptide containing a 4-iodo-L-phenylalanine resi-

due (iF-peptide, 3) was prepared as described below. 4-Iodo-L-phenylalanine was

derivatized with 9-Fluorenylmethyl chloroformate in the presence of N,N-diisoprpopyl-

ethylamine (DIEA) to afford Fmoc-4-iodo-L-phenylalanine. The iF-peptide was synthe-

sized on Fmoc-CLEAR-Amide Resin (0.1 mmol) using Fmoc chemistry. Fmoc and

side-chain protected amino acids were used in 4-fold molar excess over the N-termini.

Fmoc amino acids were activated in situ by standard reagents such as 2-(1H-benzotri-

azole-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxy-

benzotriazole (HOBt), and DIEA. The Fmoc group was removed with piperidine (20 %

(v/v)) in N,N-dimethylformamide (DMF). The peptide was cleaved and deblocked by

stirring them at RT for 6 h in trifluoroacetic acid (TFA; 8.25 mL), phenol (0.75 g),

ethane dithiol (0.25 mL), thioanisole (0.5 mL), and water (0.5 mL). The yellow-orange

crude peptide solution was filtered and the resin was washed with TFA (3 mL); the

filtrate and wash were collected in a 50-mL Falcon tube. Ice-cold diethyl ether (40 mL)

was added, and a white precipitate was immediately appeared. After centrifugation at

5000 rpm for 5 min the precipitate (the material) was washed once with ice-cold dieth-

yl ether. The resulting crude peptide was dried in vacuo and purified via preparative

ODS-HPLC (COSMOSIL® 5C-18 AR-II, 20x250 mm) using a linear gradient of 0-45%

acetonitrile/0.1% TFA. The pure iF-peptide was lyophilized to give a colorless

amorphous solid (10 mg).

Preparation of Y32-Ras-His protein: The human c-Ha-Ras protein (1-171) fused

with hexahistidine (H6) residues (Y32-Ras-His; MTEYKLVVVGAGGVGKSALTIQLIQ-

NHFVDE-Y32-DPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFL

CVFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAARTVESRQAQDLARS

YGIPYIETSAKTRQGVEDAFYTLVREIRQHKLRKL-GSLVPRGSHHHHHH) was ex-

pressed from pK7-Ras-His under control of the lac promoter in the E. coli DH5α strain.

The Y32-Ras-His protein was purified using Ni-NTA agarose (Ni nitriloacetic acid-

agarose beads) and a Superdex 75 column. The Y32-Ras-His protein (0.5 mgmL-1)

- 8 -

was sealed in a laminated aluminum bag with an oxygen absorber, and stored at

-20 °C in a solution containing Tris-HCl (50 mM, pH 7.6), NaCl (75 mM), MgCl2 (1 mM),

and glycerol (5.5 M).

Preparation of Y32-Ras protein: The wild-type Ras protein (1-171) without a hexa

-histidine (H6) tag (Y32-Ras; MTEYKLVVVGAGGVGKSALTIQLIQNHFVDE-Y32-

DPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKS

FEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSA

KTRQGVEDAFYTLVREIRQHKLRKL) was expressed from pRGH in the E. coli TG1

strain.[3] The Y32-Ras protein was purified using DEAE-Sephacel and a Superdex 75

column. The Y32-Ras protein (20 mg mL-1) was sealed in a laminated aluminum bag

with an oxygen absorber, and stored at –20 °C in a solution containing HEPES-KOH

(40 mM, pH 8.0), NaCl (80 mM), MgCl2 (0.8 mM), and glycerol (4.3 M).

Preparation of iF32-Ras protein: The H6-fused iF32-Ras protein (iF32-Ras-His;

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDE-iF32-DPTIEDSYRKQVVIDGETCLLDI

LDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPM

VLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQHKL

RKL-GSLVPRGSHHHHHH) was expressed using an E. coli cell-free translational sys-

tem. The 4-iodo-L-phenylalanyl-tRNAPheCUA was prepared as described previously.[4]

The pre-acylated tRNA was dissolved in the E. coli cell-free translation mixture[5] con-

taining pK7-Ras-His-32am, encoding the H6-fused Ras protein with a nonsense codon

TGA at position 32. The cell-free system was incubated at 30 °C for 30 min. The re-

sulting in vitro translation product was purified using Ni-NTA agarose (Ni nitriloacetic

acid-agarose beads). The bound iF32-Ras-His was eluted with Tris-HCl (50 mM, pH

7.9), NaCl (0.3 M), imidazole-HCl (0.25 M, pH 7), and Mg(OAc)2 (10 mM). The eluant

was concentrated by ultrafiltration. Ascorbic acid (final concentration 4 mM) was mixed

with the protein solution. It was stored at –80oC for the Sonogashira reaction. It was

also buffer-exchanged into HEPES-KOH (40 mM, pH 8.0) containing glycerol (4.3 M,

40%(v/v)), NaCl (80 mM), and MgCl2 (0.8 mM) for the Mizoroki-Heck reaction. The

iF32-Ras-His stock solution (96% purity)[4] was sealed in a laminated aluminum bag

with an oxygen absorber, and stored at -20oC.

Preparation of iF174-Ras protein: The H6-fused Ras protein containing 4-iodo-L-

phenylalanine at position 174 (iF174-Ras-His; MTEYKLVVVGAGGVGKSALTIQLIQ-

NHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVF

- 9 -

AINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGI

PYIETSAKTRQGVEDAFYTLVREIRQHKLRKL-DP-iF174-SHHHHHHKL) was ex-

pressed from the PCR amplified template in the cell-free translational system. Purifi-

cation and buffer exchange of the iF174-Ras-His protein was carried out in the same

manner as for iF32-Ras-His.

Preparation of biotin analogues: Vinylated biotin (1) was synthesized through a

condensation between D-(+)-biotin and 3-amino-1-propene by 1-ethyl-3-(3-dimethyl-

aminopropyl) carbodiimide (EDC) hydrochloride and DIEA. Propargylated biotin (2)

was synthesized through a condensation between D-(+)-biotin and 3-amino-1-propyne

in the same manner.

Preparation of palladium catalysts: The catalyst solution for the Mizoroki-Heck re-

action was prepared under argon or nitrogen as described below. A stock solution of

Pd(OAc)2 (20 mM) in DMSO (12.8 M) and TPPTS (20 mM) in water was mixed in a

molar ratio of 1:1 , and this was incubated for 30 min at RT before use.

The catalyst solution for the Sonogashira reaction was prepared according to the

procedure in the literature with some modifications.[6] Pd(OAc)2 (10 mM) and TPPTS

(50 mM) in water was mixed with CuOTf (20 mM) in DMSO in a volumetric ratio of 5:1.

The catalyst was sealed in a laminated aluminum bag with the oxygen absorber, and

then rotated at 4oC for five days.

Mizoroki-Heck reaction using a peptide substrate: The Mizoroki-Heck reaction

was screened using the iF-peptide (Table 1). The prereaction mixtures containing vari-

ous concentrations of iF-peptide, vinylated biotin (1), salts and bases were thoroughly

dried on a vacuum centrifuge. They were freshly resuspended in a reaction mixture

(9.5 µL) containing DMSO and BSA under nitrogen or argon in a glove box. The oxy-

gen concentration was kept below 2%. The catalyst solution (0.5 µL) was finally added

to start the Mizoroki-Heck reaction. The reaction mixtures were sealed in a laminated

aluminum bag with an oxygen absorber and incubated under various conditions. The

reaction mixtures were dried in vacuo and stored at –20 °C. These samples were dis-

solved in water (20 µL) and immediately analyzed on COSMOSIL ® 3C-18 AR-II (4.6x

100 mm) at a flow rate of 1 mL/min using a linear gradient of 0-60% acetonitrile/0.1%

TFA in 12 min. The molar absorption coefficient of the Mizoroki-Heck reaction product

(4) was 1.1 X 104 M-1cm-1 at 215 nm, while that of the substrate (3, iF-peptide) was 1.5

x 104 M-1cm-1 at 215 nm.

- 10 -

Synthesis of biotinylated peptide 4 under optimized Mizoroki-Heck reaction

conditions: Vinylated biotin (1; 10 mM, 2.5 mL), iF-peptide (3, 2 mg, 7 mM, 560 µL),

and TAPS-NaOH (2M, 1.6 mL, pH 8.3), were mixed and thoroughly dried on a vacuum

centrifuge. This mixture was freshly resuspended in DG (1.6 mM, 3.2 mL) under nitro-

gen in a glove box. The oxygen concentration was kept below 2%. The catalyst solu-

tion (0.8 mL) was finally added. The reaction mixture was sealed in a laminated alu-

minum bag with an oxygen absorber and incubated at RT for 14h. The mixture was

dried on a vacuum centrifuge and stored at –20oC. It was purified by ODS-HPLC

(COSMOSIL® 5C-18 AR-II, 10x250 mm) at a flow rate of 3 mL/min using a linear gra-

dient of 0-40% acetonitrile/0.1% TFA in 25 min to give the bF-peptide 4 (0.8 mg, 25%):

HRMS (FAB) calculated for C31H45N6O9S [(M+H)+] 677.2969, found 677.3015.

Sonogashira reaction using a peptide substrate: The Sonogashira reaction was

screened using the iF-peptide (Table 2). The prereaction mixtures containing various

concentrations of iF-peptide, propargylated biotin (2) and salts were thoroughly dried

on a vacuum centrifuge. They were freshly resuspended in a reaction mixture (9.5 µL)

containing DMSO (and proteins) under nitrogen or argon gas. The oxygen concentra-

tion was kept below 2%. The catalyst solution (0.5 µL) was finally added. The reaction

mixtures were sealed in a laminated aluminum bag with an oxygen absorber and

incubated under various conditions. The reaction mixtures were dried on a vacuum

centrifuge and stored at –20oC. These samples were dissolved in water (20 µL) and

immediately analyzed on COSMOSIL ® 5C-18 AR-II (4.6x250 mm) at a flow rate of 1

mL/min using a linear gradient of 0-100% acetonitrile/0.1% TFA in 30 min. The molar

absorption coefficient of the Sonogashira reaction product (5) was 1.3 x 104 M-1cm-1 at

215 nm, while that of the substrate (3, iF-peptide) was 1.5 x 104 M-1cm-1 at 215 nm.

Synthesis of biotinylated peptide 5 under optimized Sonogashira reaction con-

ditions: Propargylated biotin (2; 10 mM, 320 µL), iF-peptide (3, 2 mg, 10 mM, 0.4 mL),

and TAPS-NaOH (2M, 0.4 mL, pH 8.3) were mixed and thoroughly dried on a vacuum

centrifuge. Next, Triton® X100 (100% (v/v), 20 µL) was added and further dried on a

vacuum centrifuge. This mixture was freshly resuspended in DMSO in water (2.4 M,

3040 µL) under nitrogen in a glove box. The oxygen concentration was kept below 2%.

The catalyst solution (960 µL) was finally added. The reaction mixture was sealed in a

laminated aluminum bag with an oxygen absorber and incubated at RT for 90 min.

The reaction mixture was dried on a vacuum centrifuge and stored at –20 °C. This

- 11 -

mixture was purified by ODS-HPLC (COSMOSIL® 5C-18 AR-II, 10x250 mm) at a flow

rate of 3 mL/min using a linear gradient of 0-40% acetonitrile/0.1% TFA in 25 min to

give bF-peptide 5 (0.5 mg, 15%): HRMS (FAB) calculated for C31H41N6O9S [(M-H)-]

673.2656, found 673.2708.

Protein functionalization by the Mizoroki-Heck reaction: The prereaction mixture

(100 µL) containing vinylated biotin (0.4 mM), NaOAc (0.1 M), MgCl2 (8 mM), tetrabutyl-

ammonium chloride (5 mM), decyl-β-D-glucopyranoside (0.16 mM, 0.005% (w/v)), tyr-

amine-HCl (1 mM), and TAPS-NaOH (10 mM, pH 8.3) was thoroughly dried on a vacu-

um centrifuge. It was freshly dissolved in a reaction mixture (9.5 µL) containing DMSO

(1.3 M, 10%(v/v)) and iF32-Ras-His (2.85 µg) under nitrogen or argon in a glove box.

The oxygen concentration was kept below 2%. The catalyst solution (0.5 µL) was

finally added to start the Mizoroki-Heck reaction. The reaction mixture was sealed in a

laminated aluminum bag with an oxygen absorber, and this was incubated at 5 °C for

50 h. The crude reaction product was mixed well with the extraction buffer (100 µL)

containing sodium stearate (2 mM, 0.06% (w/v)), Triton® X100 (0.03% (v/v)), NaCl

(100 mM), and Tris-HCl (22 mM, pH 7.6). The precipitate was removed by centrifuga-

tion at 4 °C. The supernatant was subjected to a GST-pull down assay.

Protein functionalization by the Sonogashira reaction: The protein substrate (iF32

or iF174-Ras-His; 2 µL) was mixed with the prereaction mixture (30 µL) containing

TAPS-NaOH (120 mM, pH 8.3), Triton® X100 (0.5% (v/v)), and propargylated biotin

(20 mM) under nitrogen or argon in a glove bag. Next, the catalyst solution (8 µL) was

added to start the Sonogashira reaction. The reaction mixture was incubated at 6 °C

for 80 min.

LC-MS and LC-MS/MS analyses: LC-MS and LC-MS/MS analyses were performed

as described previously[7,8] with some modifications . The crude reaction mixture (10

µL) was mixed with the SDS gel-loading buffer (20 µL) containing DTT (200 mM), SDS

(140 mM, 4% (w/v)), glycerol (2.2 M, 20% (v/v)), bromophenol blue (0.1% (w/v)), and

Tris-HCl (100 mM, pH 6.8), immediately after the Sonogashira reaction. The sample

was incubated at 60 °C for 1 min and applied on SDS-PAGE (12% NuPAGE® Bis-Tris

gel). The 21kDa protein band was excised from the gel, and it was incubated in the

digestion buffer (50 µL) containing Lys-C (0.05 µg), decyl-β-D-glucopyranoside (1.6

mM, 0.05%(w/v)), and Tris-HCl (0.05 M, pH 9.0) at 37 °C for 12 h. The liberated pep-

tides were separated on Mightysil® C18 (1 x 50 mm) at a flow rate of 30 µL/min using

- 12 -

a linear gradient of 2-60% solvent B in 40 min, where solvent A and B consisted of

0.09% (v/v) TFA in water, and 0.075% (v/v) TFA and 80% (v/v) acetonitrile in water,

respectively. The eluant was analyzed with a LCQ ion-trap mass spectrometer with a

house-made ESI probe.

GST pull-down assay: An in vitro binding assay was carried out as described below.

A tenfold volume of the extraction buffer containing sodium stearate (2 mM, 0.06%

(w/v)), Triton X100 (0.03% (v/v)), NaCl (20 mM), and Tris-HCl (22 mM, pH 7.6), was

added the reaction mixture. The precipitate was removed by centrifugation at 4 °C.

Next, the supernatant was mixed with N,N'-dimethylthiourea (50 mM), BSA (0.5

mg mL-1) and a non-hydrolyzable GTP analogue (GTPγS; 0.1 mM) or GDP (0.1 mM).

This was incubated at RT for 10 min, and mixed with MgCl2 (10 mM), GST-Raf-1-RBD

(1 µg) and glutathione-Sepharose® 4B beads suspended in water (20 µL), then was

incubated on ice for 1h. The resin was washed with the washing buffer (1 mL) con-

taining MgCl2 (5 mM), NaCl (20 mM), Tris-HCl (20 mM, pH 7.6) and N,N'-dimethyl-

thiourea (50 mM). The bound proteins were then separated on SDS-PAGE.

XRF analysis: The amount of transition-metal ions bound on proteins was estimated

by XRF analysis as described below. The wild-type Ras protein (2 mg, 1 mL) was in-

cubated at RT for 1h under nitrogen in the presence of Pd(OAc)2 (2 mM), TPPTS (10

mM), CuOTf (0.8 mM), DMSO (2.3 M), TAPS-NaOH (200 mM, pH 8.3), Triton® X100

(0.4% (v/v)), HEPES-KOH (4 mM, pH 8.0), NaCl (8 mM), MgCl2 (0.1 mM), and glycerol

(0.4 M). N,N'-dimethylthiourea (1 M, 56 µL) was added, and this mixture was applied

onto a PD-10 column that had been equilibrated with a desalting buffer containing

Tris-HCl (10 mM, pH 7.6), NaCl (155 mM), MgCl2 (2 mM), and MeOH (0.6 M). Further-

more, the desalting buffer (2 mL) was applied onto the PD-10 column and the sample

(3.1 mL) was collected in a tube. The concentration of the Ras protein was determined

by the Bradford method using BSA as a standard. The recovered Ras protein (82 µg,

3 mL) was lyophilized and dissolved in nitric acid (2 M, 5 mL). A known amount of

cobalt ion (10 ppm) was mixed as an internal standard. This sample was spotted on a

filter, dried under a heat lamp, and subjected to XRF analysis using MoKα radiation

(17.5 keV). The amount of palladium and copper ions was estimated by measuring

peak areas against the Co-Kα peak at 6.93 keV, assuming that the ratio of the Pd-Lα,

Co-Kα, and Cu-Kα peak areas are 1:27.5:39 when they are at the same concentration.

The lower detection limit is 0.004 ppm (60 nM; copper).

- 13 -

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